表达反义hREV3基因片段的真核细胞表达质粒的构建

目的：构建真核细胞表达重组体，为今后利用反义核酸阻断技术研究ｈＲＥＶ３基因功能４及其与细胞遗传不稳定性的关系提供物质材料。方法：用内切酶ＢａｍＨⅠ及Ｂａｍ ＨＩ＋ＨｉｎｄⅢ分别对ｐＢＳ－ｈＲＥＶ３进行单酶切和双酶切，从而得到含ｈＲＥＶ３ ｃＤＮＡ特异性保守序列的两种长度片段，分别将两者插入真核细胞载体；ｐＢＫ－ＲＳＶ和ｐＭＡＭｎｅｏ ａｍｐ＾－。

Construction of eukaryotic expression plasmid expressing antisense fregment of hREV3

AIM: To construct the eukaryotic expression plasmid to study the role of hREV3 gene in genesis of genetic instability by means of antisense technology. METHODS: Two fragments with different length (742bp and 357bp) that constain the specific conserved region of hREV3 cDNA were obtained by digesting plasmid pBS-hREV3 with restriction enzymes BamH I and BamH I plus Hind III respectively .Two kinds of fragments were inserted into two kinds of eukaryotic expression vector : pBS-RSV(used for constant expression and pMAMneo amp -(used for inducible expression).The inserting orientation of recombinants were identified by digesting with restriction enzymes and electrophoresis analysis. RESULTS: Obtained five kinds of recombinant containing the insertions in sense and antisense orientation. CONCLUSION: These results provide an important basis to further study in genetic instability.